Secret Parameter in Flow Cytometry - Time Application Sharing

In this edition of the flow cytometry guide, we will mainly discuss a parameter that is crucial in Flow Cytometry experiments but often overlooked - Time.

What is Time?

Time refers to the "time" parameter, which is typically used in conjunction with other parameters to reflect real-time changes during the experimental process. This provides us with a wealth of information, allowing us to obtain more accurate and comprehensive analytical results during the experiment.

Specific Applications of the Time Parameter:

Firstly, it is used in the detection of fluid stability. We believe that everyone is aware of the importance of fluid stability in flow cytometry experiments. It is essential to pay attention to the stability of the fluid flow during the experiment. If the fluid flow is unstable, the flow cytometry results will be poor, and there may be a decrease in instrument resolution, an increase in CV values, or even loss of detection signals. Since fluid stability is so crucial, how can we observe whether the fluid flow is stable? This is where the combination of quantity and time gates comes into play.

Figure 1: flow cytogram of stable fluid flow

As shown in the figure, by selecting Time as the x-axis and Count as the y-axis, we can observe the real-time speed of sample acquisition. Under normal circumstances, the sample acquisition speed should remain relatively consistent.

Figure 2: Flow cytogram of peristaltic pump tube aging

As shown in Figure 2, by selecting Time as the x-axis and Count as the y-axis, we can observe the real-time speed of sample acquisition. In the illustrated results, we can clearly see that the sample flow is highly unstable, with varying speeds, and there are periods when no sample is acquired. This is highly detrimental to data collection, and if such results are encountered during the experiment, caution must be exercised, and if necessary, the sample should be reanalyzed.

Fluid instability can have various causes, such as pipeline blockage or aging of the peristaltic pump tube. By further analyzing the Time graph, we can identify the specific causes of fluid instability. In Figure 2, we can observe that the fluid flow changes in a clearly periodic pattern, indicating aging of a specific segment of the peristaltic pump tube. Each time the sample passes through this section of the tube, there is a failure to acquire the sample, indicating the need to replace the peristaltic pump tube.

If it is a pipeline blockage, the Time graph will likely fall into the following two scenarios:

Figure 3: Transient blockage of the pipeline flow cytogram

Figure 4: Permanent blockage of the pipeline flow cytogram

In Figure 3, we can see that after a long period of time without sample acquisition, there is a sudden increase in sample acquisition speed. This is often due to the sample blocking the pipeline, preventing sample acquisition. As the sample acquisition continues, the pressure inside the pipeline increases. When a certain pressure is reached, the blocked section suddenly clears, allowing a large number of cells to pass through and resulting in a rapid increase in sample acquisition speed. Of course, it is also possible to encounter more severe blockages that ultimately lead to complete pipeline occlusion (as shown in Figure 4). Therefore, when encountering a blockage in the instrument, it is essential to stop sample acquisition promptly, perform appropriate cleaning, and check for sample clumping or excessively high cell density. Choose the appropriate mesh filter or dilute the sample accordingly.

Of course, the application of Time goes beyond determining fluid stability. When combined with fluorescence channels, it can help identify unreasonable data caused by instrument instability, residual samples from the previous tube, and other unexpected situations during the experiment. By gating out this portion of the data, more reliable and accurate results can be obtained (as shown in Figure 5, the fluorescence signal in the blue box is significantly different from the overall sample and should be excluded in the actual analysis).

Figure 5: Flow cytogram with Time gate and fluorescence channel gating

Furthermore, when detecting indicators that rapidly change with time, such as expression levels, Time can be used to observe the real-time changes of the indicators.

If you have any other questions regarding the application of the Time parameter during practical experiments, please feel free to leave a comment below.